Background: Dermatophytes are group of fungi that infect keratinized tissues of human and animals. The group consist of three different genera namely, Trichophyton, Microsporum, Epidermophyton and several species within each genera. Among Trichophyton, Trichophyton rubrum is predominant, followed by various strains of Trichophyton mentagrophytes, which include both anthropophiles and zoophiles. Prevalence of dermatophytes varies with location and environmental condition. The infection is common worldwide with higher prevalence in tropical countries like India. Molecular diagnosis renders accurate identification of clinical dermatophyte isolates to species level. The main objective of this study was to determine the prevalence of dermatophytoses, isolate and identify the dermatophyte from samples of clinically suspected cases attending tertiary care centre using conventional and molecular methods. Methods: A total of 210 patients showing lesions typical of dermatophytes infection from outpatient Department of dermatology were sent to mycology unit, Department of Microbiology for the period of April 2011-March 2014 were studied. Diagnosis was confirmed by conventional and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) technique. Results: Out of 210 samples received, tinea corporis was the predominant clinical site which was followed by tinea cruris. A total of 143 dermatophytes were isolated from the clinical samples. T. rubrum was the predominant etiological agent with 70/143 isolates and T. mentagrophytes was the second most common with 64/143 isolates. Amplification of internal transcribed spacers (ITS) was successful in all the clinical isolates by PCR and produced species specific banding pattern in RFLP using restriction enzyme Mva I. Conclusions: Among dermatophytoses, T. rubrum was the predominant etiological agent present in the whole of Chennai District and T. mentagrophytes takes the second place.Key words: Molecular speciation, Internal transcribed spacers, Mva I, Trichophyton rubrum, Trichophyton mentagrophytes, PCR-RFLP